System and method for cross-talk cancellation in a multilane fluorescence detector

ABSTRACT

The present invention is directed to a system and method for cross-talk cancellation for multi-lane fluorescence detectors. The invention may be implemented in accordance with a variety of systems, including systems for multi-capillary electrophoresis. The present invention is based on a special calibration procedure for determination of a channel cross-talk matrix and enables an accurate separation of the fluorescence emitted from individual capillary lanes. The proposed method for cross-talk calibration and removal is very useful for design and development of multi-lane single photon counting detection systems.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of International ApplicationPCT/US05/27059, filed Jul. 29, 2005, which claims priority to U.S.Provisional Patent Application 60/592,170, filed on Jul. 29, 2004, whichare hereby incorporated by reference in their entireties.

FIELD OF THE INVENTION

The present invention relates generally to photodetection systems andmethods. More specifically, the present invention is directed to asystem and method for cross-talk cancellation for multilane fluorescencedetectors, such as those used in capillary electrophoresis for DNAsequencing.

BACKGROUND OF THE INVENTION

Capillary electrophoresis is a widely-used technique in high-throughputDNA sequencing. In practically all DNA sequencers, fluorescence labelingtechniques are used for sequence detection. A number of extremelysensitive fluorescence detection techniques are available based onregistering single photons, commonly referred to as the single photondetection (SPD) techniques. Such techniques are known in the prior artand are described in G. Papageorgas, H. Winter, H. Albrecht, et al.,IEEE Trans. on Instrumentation and Measurement, 1999, Vol. 48, 6, pp.1166-1177; Y. E. Tiberg and V. N. Paulauskas, Instruments andExperimental Techniques, 1981, Vol. 23, 5, pp. 1252-1255, A. M.Evtyushenkov, Y. F. Kiyachenko, G. I. Olefirenko and I. K. Yudlin,Instruments and Experimental Techniques, 1982, Vol. 24, 5, pp.1265-1267, K. D. Shelevoi, Instruments and Experimental Techniques,1985, Vol. 28, 3, pp. 614-616, the texts of which are fully incorporatedherein by reference. Because of their complexity and cost, SPDtechniques were mostly used for specialized scientific applications, astime resolved fluorescence spectroscopy or detection of singlefluorescent molecules.

Recently, SPD techniques have been employed in single-lane DNAsequencing instruments, as documented in L. Alaverdian, S. Alaverdian,O. Bilenko, et al., I. Bogdanov, S. Domrachev, E. Filippova, D.Gavrilov, B. Gorbovitski, M. Gouzman, I. Gudkov, N. Lifshitz, S. Luryi,V. Ruskovoloshin, A. Stepukhovich, M. Tcherevishnik, G. Tyshko and V.Gorfinkel, Abstract Book of the conference “Advances in Genome Biologyand Technology”, p. 47, Marco Island, Fla., USA, Feb. 3-6, 2001; and L.Alaverdian, S. Alaverdian, O. Bilenko, I. Bogdanov, E. Filippova, D.Gavrilov, B. Gorbovitski, M. Gouzman, G. Gudkov, S. Domratchev, S.Kosobokova, N. Lifshitz, S. Luryi, V. Ruskovoloshin, A. Stepoukhovitch,M. Tcherevishnick, G. Tyshko, V. Gorfinkel, Electrophoresis 2002, 23,pp. 2804-2817 (hereinafter “Alaverdian et al.”), the texts of which arefully incorporated herein by reference.

However, one of the most difficult challenges in the development of suchsystems is the elimination of the lane cross-talk caused by both opticaland electronic cross-talk phenomena between channels of the singlephoton detector. The prior art systems have not addressed this issue.

There are two general types of lane cross-talk in single photondetection systems: optical and electronic. Electronic cross-talk may becaused by any multi-channel electronic module of a detection system,specifically as a result of, e.g., certain features of the electronicoptics inside of a photo-multiplying tube. Optical cross-talk may becaused by, e.g., poor quality of a capillary array image on thereceiving area of the photodetector, a contradiction betweenrequirements of certain image magnification (from a lens) necessary forprojection of the array as a whole onto the photodetector, andadditional magnification of inner capillary volumes caused by capillarywalls, a misalignment of the optical system after the capillary arrayplacement, etc.

Cross-talk can be an especially significant problem in certainphotodetection applications, such as, e.g., in DNA sequencingapplications. Different lanes of a multi-lane DNA sequencer can haveorders of magnitude variation in amplitude of fluorescent peaks.Accordingly, even very small lane cross-talk may cause ambiguity in dataanalysis.

Ultimately, some sources of channel cross-talk in a single photondetection system cannot be eliminated. Certain measures may beundertaken to reduce cross-talk, but these solutions are generally lessefficient, more complex and expensive.

In prior art systems and methods, the general approach is to eliminatecross-talk, both optical and electrical, in the system. On the opticalside, several strategies for removal of cross-talk have been employed.Examples include using an aperture mask to remove cross-talk at theinput of the multi-capillary system, decreasing the distance of thecollection system, and employing a smaller collection angle. However,while these measures may reduce or eliminate frond-end cross-talk, theyalso reduce the light collection efficiency. As a result, thesecross-talk avoidance methods are limited in the types of equipment theymay utilize, e.g., they may employ photodetectors which do not introducecross-talk (even though these are typically the more sensitive andpowerful photodetectors).

Accordingly, it is an object of the present invention to provide a novelsystem and method for calibration and elimination of channel cross-talkto enable an accurate separation of fluorescence signals emitted byindividual capillaries.

SUMMARY OF THE INVENTION

In accordance with an exemplary embodiment of the present invention, amethod is provided for reducing cross-talk in a multi-channelphotodetection system, including the steps of determining a cross-talkmatrix for the system; operating the system to detect data, and applyingthe cross-talk matrix to the detected data to reduce or remove channelcross-talk in the detected data.

In another exemplary embodiment of the present invention, a system isprovided for reducing cross-talk in a photodetection system, the systemincluding at least a processor and a memory, which memory storesinstructions that may cause the processor to perform the steps ofdetermining a cross-talk matrix, operating the system to obtainfluorescence data, and applying the cross-talk matrix to thefluorescence data to reduce cross-talk in the fluorescence data.

In another exemplary embodiment of the present invention, a system isprovided for multi-capillary electrophoresis, the system including atleast a light source, at least one multi capillary array positioned toreceive light from the light source and filled with a material whichproduces a useful radiation caused by illumination with said lasersource, a device for separating and identifying more than one spectralband of said useful radiation, a multi channel photodetector positionedto receive said useful radiation from the multi capillary array, aprocessor configured to receive data originating from the photodetectorand, a memory coupled to the processor and containing instructionswhich, when executed by the processor, configure the processor toperform steps including determining a cross-talk matrix, operating thesystem to detect fluorescence data, and applying the cross-talk matrixto the fluorescence data to reduce cross-talk in the fluorescence data.

One of the important benefits of the present invention is that a muchlarger variety of equipment may be employed in designing multi-capillarysystems. A larger collection angle may be utilized to provide a highercollection efficiency. Photodetectors that are more sensitive may beemployed, even if they do introduce cross-talk into the system.Additionally, the light collection efficiency for a system according tothe present invention may be much higher than in prior art systems. Theapproach thereby provides a system designer with substantially greaterfreedom in the selection of system components.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a block diagram illustrating an exemplary system according tothe present invention; and

FIG. 2 is a flow diagram of a method in accordance with an exemplaryembodiment of the present invention.

Throughout the Figures the same reference numerals and characters,unless otherwise stated, are used to denote like features, elements,components or portions of the illustrated embodiments. Moreover, whilethe present invention will now be described in detail with reference tothe Figures, it is done so in connection with the illustrativeembodiments.

DETAILED DESCRIPTION OF THE INVENTION

Referring now to FIG. 1, an exemplary embodiment of a multi-lane DNAsequencing system according to the present invention is shown. In thisexemplary embodiment, the system of FIG. 1 may be similar to the systemdescribed in Alaverdian et al. in with the exception of the novel lanecross-talk removal function implemented in accordance with the presentinvention.

The exemplary embodiment of FIG. 1 may include a multi-capillary array12. In a preferred embodiment the array may be a 16- or 48-capillaryarray, such as those made available by Applied Biosystems, Inc., or maybe any other multi-capillary array. The multi-capillary array 12 maypreferably be inserted into a precision holder.

In accordance with the exemplary embodiment of FIG. 1, a laser beamilluminates the array from the side and excites fluorescence in thecapillaries of the array 12. In an exemplary embodiment, the laser beammay be generated by a fiberized, single mode 25 mW NdYAG laser (532 mm),such as those available from Crystal Lasers, Inc. In order to illuminatethe multi-capillary array from the side and excite fluorescence in allchannels with high efficiency, an optical system comprising a collimatorand microscope objective (JIS 10×/0.25) may be employed to reduce thebeam size to a size of, e.g., 40 microns.

The fluorescence excited in the multi-capillary array is captured by alens of a high numerical aperture, passed through a spectral separatingdevice 13, and projected onto photosensitive pixels of a photodetector14. In a preferred embodiment, the spectral separating device 13 may bea rotating filter wheel comprising four band pass filters passing thefluorescence in four different spectral bands. Such a device may beadvantageously employed because the system cross-talk effects may differin different spectral bands. The spectral separator device may isolateparticular spectral bands for cross-talk analysis. Revolutions of thefilter wheel may be synchronized with the photodetector 14 so that thephotodetector is provided with information regarding what color light itis receiving at any given time. Although outside the scope of thepresent invention, various other optical filtering may be employed toensure efficient transmission of the fluorescence excited in themulti-capillary array to the photodetector 14. For example, the lens maypreferably be any lens with high numerical aperture, such as thecommercially-available Canon EF 50 mm 1:1.4. The photodetector 14 maypreferably be a 32-channel PMT, such as the H72060 developed byHamamatsu. However, any photodetector and filtering apparatus may beemployed in accordance with the present invention, as would beunderstood by one of ordinary skill in the art.

The photodetector 14 may preferably be operated in a single photoncounting mode in accordance with this exemplary embodiment of thepresent invention. Each channel of photodetector 14 produces an outputstream of short pulses in response to an input incident photon flux. Theoutput pulses may generally range between 0.4-0.6 mA with correspondingpeak voltage between 8 and 12 mV. In order to facilitate photon countingdetection mode, a pulse amplifier and photon counter 16 may be employed.These functions may be performed using a single piece of hardware orseveral pieces of hardware, depending upon the particulars of theembodiment. For example, a 32-channel pulse amplifier and a 32-channelphoton counter may be employed in accordance with the preferredembodiment.

The amplifier comprises 32 identical pulse amplifying channels with gainof 35-40 dB, bandwidth of 0.5-4,000 MHz and 32 fast comparators. Thesecomparators have a rise and fall time of about 2 ns, which limits theminimum pulse width to approximately 4.5 ns. After amplification, adigitization (counting) of the amplified signal is performed by a homedesigned 32-channel photon counter. In order to synchronize the counterwith the rotating 4-color filter wheel and to identify band-passfilters, we use a 2-bit Gray code from two sensors installed on thefilter wheel. The change of the code word at the sensor outputsindicates the change of the filter in alignment. The counting of theinput photon pulses in each of the 32 channels is performed by summationof pulses arriving to the channel input during the time intervals when aparticular band-pass filter is in alignment with the photodetector.

Data collected by the counter is transferred to a computer device suchas a personal computer using standard IEEE 1284 Parallel Port Interfaceor other standard computer interface. The data is transferred in samplesusing a binary format. One data sample is collected during one fullrevolution of the filter wheel. The frame consists of count valuesobtained in 4 fluorescence detection bands (4 color filters) for each ofthe 32 detection channels. The frames are sent in order of theirgeneration, determined by the filter code on the wheel and the directionof its rotation. Each frame starts with a 6-byte header which includesthe following fields: 1-byte counter type, 2-byte frame number, 1-bytecolor code (filter number) and a 2-byte counting period length. Theframe number contains the number of the current frame. The number isincremented by 1 for each following frame thus forming a rising sequencewith overflow. The frame numbers serve as synchronization marks and areused by the data processing software to find data frames in a continuousdata stream. Frame numbers are also used for verification of dataintegrity and for finding errors introduced by interference in thetransmission line. The duration of the counting period is measured inmilliseconds and is represented by a 2-byte value. The time durationwhen the filter is “on” is measured separately for each filter and isused by processing software to calculate a photocount rate. The framesize is 105 bytes. During the normal operation of the system, when thefilter wheel performs ˜10 revolutions per second, the counter producesabout 4200 bytes per second, which results in approximately 1.48 MB perhour.

A special software package operating on a personal computer or othercomputing device may perform the recording and the on-line visualizationof the data transferred by the counter. The recorded data may preferablyundergo a preprocessing which includes non-linearity compensation,smoothing and lane cross-talk removal. Further processing may include anautomated base calling and assigning quality factor based on PHREDapproach.

Referring now to FIG. 2, a flow diagram of an exemplary embodiment ofthe present invention is shown. As shown in FIG. 2, a first step 20 inthe exemplary method is to determine a cross-talk matrix for a givensystem. Next, in step 22, the system is operated and fluorescence datarecorded. Finally, in step 24, the cross-talk matrix is applied to therecorded test data to remove/eliminate cross-talk and leave only thecorrect fluorescence data measurements for each photodetector channel.

Notably, the details of all processing steps in accordance with thepresent invention are similar to those described in Alaverdian et al.with the exception of the novel cross-talk removal steps. One of thecritical steps in the method according to the present invention is thedetermination of a cross-talk matrix for a given system, whichcross-talk matrix may be used to remove unwanted cross-talk componentsfrom output data. Exemplary methods for determining a cross-talk matrixin accordance with the present invention are described herein below.

The proposed method of the cross-talk removal is based on the assumptionthat the fluorescence measurement system is linear, i.e., a photocountrate registered in the n-th channel of the photodetector is a sum ofcomponents contributed by signals in all other system channels andratios of contributions from individual channels do not depend on thephotocount rate. Linearity of optical cross talk is obvious as long asthe measured photocount rate stays within the linear range of the PMT.(As described in D. Gavrilov, B. Gorbovitski, M. Gouzman, G. Gudkov, A.Stepoukhovitch, V. Ruskovoloshin, A. Tsuprik, G. Tyshko, O. Bilenko, O.Kosobokova, S. Luryi, V. Gorfinkel, Electrophoresis 2003, 24, 1184-1192(hereinafter “Gavrilov et al.”).

An exemplary system according to the present invention may haveN-channel PMT and M detection lanes. The number of detection lanes maybe determined by the number of active capillaries M in the capillaryarray (M<N). The vector of fluorescence intensities in the capillarylanes may be denoted as f=(f1, f2 . . . f_(M))^(T) and the vector ofregistered photocount rates as s=(s₁,s₂ . . . s_(N))^(T), where (.)^(T)denotes the matrix transpose. The model of the system establishesrelationship between f and s vectors:s=γ(εβCf+ω),where γ(.) is an operator accounting for non-linearity of the PMT, ε isquantum efficiency of the PMT, β is an efficiency of the optical system,C_((N×M)) is the cross-talk matrix and ω_((N×1)) is the noise vector,which defines the stochastic component of s, caused by random arrivaltimes of individual photons and distributed according to Poisson. Thequantum efficiency ε determines the percentage of photons that cause thePMT to produce a response on its output. This parameter depends on thewavelength of the fluorescent emission and in our wavelength range itstays below 7%. The efficiency of the optical system β is a parametercombining efficiency of the fluorescence collection and deliverysystems. In order to simplify the equations, a photocount rate may bedefined as a number of photons per sampling interval.

In linear approximation, the cross-talk matrix C_((N×M)) combines theelectronic and optical cross-talk in the system:C=C^(el)C^(opt).

Electronic Cross-talk

Given the same level of electronic cross-talk between the adjacentchannels of the PMT, the matrix C^(el) _((N×M)) may take the form:

$c^{el} = {\begin{pmatrix}c_{1}^{el} & c_{2}^{el} & \; & \; & \; \\c_{2}^{el} & c_{1}^{el} & c_{2}^{el} & \; & \; \\\; & c_{2}^{el} & c_{1}^{el} & ⋰ & \; \\\; & \; & ⋰ & ⋰ & c_{2}^{el} \\\; & \; & \; & c_{2}^{el} & c_{1}^{el}\end{pmatrix}.}$

Exemplary Cross-talk Matrix 1

In the above Exemplary Cross-talk Matrix 1, the only diagonalscontaining non-zero elements are the main and two adjacent diagonals,meaning that no electronic cross-talk exists between non-adjacentchannels of the PMT. Measurement results confirm that this matrix isvalid for analysis of an exemplary system employing the H7260 devicedescribed in accordance with FIG. 1. Indeed, the elements of the C^(el)are c₁ ^(el)=0.94 and c₂ ^(el)=0.03, (so that c₁ ^(el)+c₂ ^(el)=1), andthe cross talk level between non-adjacent channels stays negligiblysmall (below 0.5%).

Optical Cross Talk

Elements of the optical cross-talk matrix C^(opt) _((N×M)) depend on theproperties of the optical system. In an exemplary embodiment of thepresent invention, an optical system may include a number of capillariesin the array (number of lanes) equal to the number of channels of thephotodetector (M=N), such that C_(opt) is a square matrix of the form:

$c^{opt} = \begin{pmatrix}c_{11}^{opt} & c_{12}^{opt} & {\; c_{13}^{opt}} & \; & {c_{1,N}^{opt}\;} \\c_{21}^{opt} & c_{22}^{opt} & c_{23}^{opt} & \; & {c_{2,N}^{opt}\;} \\{c_{31}^{opt}\;} & c_{32}^{opt} & c_{33}^{opt} & \; & {\; c_{3,N}^{opt}} \\\; & \; & \; & ⋰ & \; \\{c_{N\; 1}^{opt}\;} & {c_{N,2}^{opt}\;} & {\; c_{N,3}^{opt}} & \; & c_{N,N}^{opt}\end{pmatrix}$

Exemplary Cross-talk Matrix 2

Each column of C^(opt) reflects distribution of the fluorescenceintensity collected from the corresponding capillary among all Nchannels of the PMT. The matrix may be normalized as follows:

${{\sum\limits_{i = 1}^{N}c_{ij}^{opt}} = 1};{j = {1\mspace{11mu}\ldots\mspace{11mu}{N.}}}$

If the number of lanes is smaller than the number of photodetectionchannels (M<N) and each lane is assigned to its unique primary PMTchannel, i.e., the channel to which the most of its fluorescence isforwarded, then the (N×M) matrix is constricted from ExemplaryCross-talk Matrix 2 above by removing the columns with numbers of thePMT channels that do not serve as primary.

Because estimation of the absolute value of the fluorescence intensityis not important in this analysis of the recorded sequencing traces, butrather the relative peak amplitudes are the important consideration, itis convenient to use a vector of ‘true’ photocount rate r=(r₁, r₂ . . .r_(M))^(T), which represents the rate that would be obtained in anidealized system, equipped with the ideally linear M-channel estimatorof the photocount rate and optical projection system with no cross-talk:r=εβf.

According to the above equations, the registered photocount rate s maybe related to the ‘true’ photocount rate ass=γ(Cr+ω).

The simple minimum variance unbiased (MVU) estimator for r may be foundin S. M. Kay, Fundamentals of Statistical Signal Processing. EstimationTheory. Prentice-Hall, N.J., 1993, 597 p., under assumption that theelements of the noise vector in the system are independent, identicallydistributed random values with Gaussian distribution N(0, σ):

-   -   where {circumflex over (r)} denotes the estimate of r, and        γ⁻¹(.) is the non-linearity compensation operator applied to the        elements of the vector s. The methods for        {circumflex over (r)}=(C ^(γ) C)⁻¹ C ^(γ)γ⁻¹(s),        characterization and compensation of non-linearity of a single        photon detector are described in detail in D. Gavrilov, B.        Gorbovitski, M. Gouzman, G. Gudkov, A. Stepoukhovitch, V.        Ruskovoloshin, A. Tsuprik, G. Tyshko, O. Bilenko, O.        Kosobokova, S. Luryi, V. Gorfinkel, Electrophoresis 2003, 24,        1184-1192, which is incorporated herein by reference in its        entirety. If the number of lanes M in the system is equal to the        number of photodetection channels, then C is a square (N×N)        matrix, and the equation above may be reduced to:        {circumflex over (r)}=C ⁻¹γ⁻¹(s).

Therefore, in order to find vector {circumflex over (r)}, informationabout the cross-talk matrix C must be deduced.

In order to evaluate optical cross-talk, certain assumptions about thecharacteristics of the optical system which cause the cross talk may bemade. A first assumption may be that the optical cross talk in thesystem is mainly caused by edge aberrations of the lens. Therefore, thecross-talk will depend on the fluorescence collection angle.

In an exemplary embodiment of the present invention, a system using asmaller collection angle may be referred to as Narrow Collection Angle(NCA) system, and a system using a bigger collection angle may bereferred to as a Wide Collection Angle (WCA) system. In an NCA systemthe whole fluorescence collected from each capillary is projected onlyon the effective space of the corresponding channel of the PMT. The WCAsystem can not be made to produce a sharp image of the entire capillaryarray. In an exemplary system according to the present invention, theprojected images of the central capillaries of the array may be sharpand may become more and more blurred as the detector is moved closer andcloser to the array ends. As a result of blurring, more and morefluorescence is delivered to the nearest neighbors of the designatedchannels, leading to optical cross-talk between the channels.

Experiments with fluorescence collection using variable aperture showthat the NCA approximation for an exemplary optical system is valid whenthe light collection angle does not exceed 20°. Therefore, the WCAapproximation will span all collection angles 20°<θ<θ_(max), whereθ_(max) is defined either by the lens collection angle or limited to acritical angle of total internal reflection θ_(C).

According to Snell's law,θ_(C)=sin⁻¹(n _(air) /n _(glass))

where n_(air) and n_(glass) are refractive indices of air and glasscorrespondingly (n_(air)=1, n_(glass)=1.5 , θ_(C)=41.8°). In the opticalsystem employed in accordance with an exemplary embodiment of thepresent invention, the lens collection angle is ˜45°. Depending on thetype of capillary array employed, a different equation may be applied.For example, for capillary arrays which do not include an outer glassbox, the collection efficiency may be expressed as:

$\beta_{\max} = {{\frac{4\; n_{glass}n_{air}}{\left( {n_{glass} + n_{air}} \right)^{2}}\left\lbrack {1 - {\cos\left( \theta_{\max} \right)}} \right\rbrack} = {0.28.}}$

For capillary arrays of the type which are immersed into a glass quvettefilled with a refractive index liquid, the collection efficiency may bemodified and expressed as:

$\beta_{C} = {{\frac{4\; n_{glass}n_{air}}{\left( {n_{glass} + n_{air}} \right)^{2}}\left\lbrack {1 - {\cos\left( \theta_{c} \right)}} \right\rbrack} \approx 0.25}$

Thus, collection efficiencies for both capillary array types are veryclose. Optical parameter β may be denoted as β^(NCA) and β^(WCA) for NCAand WCA systems correspondingly. Assuming a maximum value ofβ_(max)≈0.25 and that maximum value of β^(NCA) is β^(NCA)(20°)=0.058,the following may be shown:β^(WCA)≈4β^(NCA)

A lane cross-talk removal method in accordance with the presentinvention may be implemented to remove cross-talk from an NCA system.The NCA system that provides ideally focused image may be described bythe diagonal matrix:C ^(opt) =diag(1,1 . . . 1).

Therefore for the NCA system C=C^(el). Due to stability of theelectronic cross-talk matrix C^(el), the matrix C can be computed oncefor the PMT and used until the PMT is replaced.

A lane cross-talk removal method in accordance with the presentinvention may be implemented to remove cross-talk from a WCA system. Inthe WCA system, depending on the image quality, the Exemplary Cross-talkMatrix 2 may have non-zero elements on the main and several adjacentdiagonals. Analytical evaluation of the matrix C for the WCA systemrequires evaluation of the C^(opt), which depends on specific design ofthe optical system and may vary not only from system to system, but evenafter every replacement of the multi-capillary array because of thesystem misalignment caused by a finite tolerance of the arraypositioning system.

Therefore, for determining elements of the C matrix for specificdetection system, it is more practical to use a special calibrationprocedure based on sequencing data obtained from the system calibrationexperiment. Obviously, the system calibration must be performed eachtime a new capillary array is installed.

The calibration of a cross-talk cancellation algorithm in accordancewith an exemplary embodiment of the present invention includesestimation of the cross-talk matrix. Two approaches may be employed inaccordance with an exemplary embodiment of the present invention—staticand dynamic approaches to calibration.

Static Approach

This approach to calibration is well suited for photodetection units.Static calibration may be performed at the stage of device manufacturingbefore the photodetection unit is installed in the system. The n-thcolumn of a cross-talk matrix is obtained by illuminating the n-th pixel(channel) of the photodetector and recording responses at the outputs ofeach channel. For example, in calibration of a linear array PMT, theillumination of a single channel can be achieved using a low aperturesingle mode fiber. The fiber may be equipped with a tip lens to narrowthe aperture. Before each experiment the background noise levels may berecorded in each channel. The background levels are subtracted from theresponses before their values are used to form the columns of across-talk matrix.

Dynamic Approach

The dynamic approach may preferably be employed for calibration of adetection system as a whole. The resulting matrix from such calibrationcombines the internal and external (i.e., electronic and optical)cross-talk. In this approach, the cross-talk matrix is estimated fromthe results of the experiment. An algorithm for matrix estimation isutilized to extract the cross-talk information from the recorded dataset using knowledge about the specific conditions of the experiment.This approach to calibration of the cross-talk cancellation algorithmmay be applied in an exemplary fluorescence detection system formulti-capillary electrophoresis in accordance with the presentinvention.

Estimation of the lane cross talk includes execution of a series ofshort sequencing runs. The number of runs in the series is determinedexperimentally as a number of capillaries between two closest lanes withnegligibly small cross-talk.

In one exemplary embodiment of the present invention, for calibration ofthe lane cross-talk an internal lane standard (ILS-600, Promega Corp)may be used. This sample produces a number of well separated peaks inthe 580 nm-620 nm wavelength range on a rather low background level(˜1,000 count/s) which generates very low photon counting noise (<250c/s). Thus, since the typical count rate of the detected ILS peaksvaries in the range of ˜100,000 c/s-50,000 c/s, the system may becalibrated for as low as 1% lane cross-talk with signal-to-noise ratiolarger than 1.

In a case when cross-talk affects only adjacent channels, thecalibration experiment consists of only two short runs during which theILS-600 sample is loaded in either odd or even capillaries of the array.Both recorded data sets may also undergo smoothing and backgroundremoval. The odd columns of C may be found using the ‘odd’ data set andthe even columns using the ‘even’ data set. In both data sets the highquality peaks may be detected in each channel that has a loadedcapillary assigned to it during the corresponding sequencing run. If Jpeaks are detected in the channel n_(i) to which the fluorescence fromthe i-th lane is primarily projected, then:

${{b_{1} = {\frac{1}{J}{\sum\limits_{j = 1}^{J}\frac{A_{n_{i} - 1}\lbrack j\rbrack}{A_{n_{i}}\lbrack j\rbrack}}}};{b_{2} = {\frac{1}{J}{\sum\limits_{j = 1}^{J}\frac{A_{n_{i} + 1}\lbrack j\rbrack}{A_{n_{i}}\lbrack j\rbrack}}}}},$where A_(ni)[j] is the height of the j-th peak in the channel n_(i) andA_(ni−1)[j] and A_(ni+1)[j] are the heights of the cross-talk inducedpeaks in the adjacent channels. The height of the peaks may bedetermined from the data set obtained by removal of baseline from theraw experimental data. The baseline removal is an important step,because the baseline may constitute significant fraction of the recordedraw data value. Then the non-zero elements of the i-th column of C maybe expressed as:

${c_{n,i} = \frac{1}{1 + b_{1} + b_{2}}};{c_{{({n_{i} - 1})}i} = \frac{b_{1}}{1 + b_{1} + b_{2}}};{c_{{({n_{i} + 1})}i} = {\frac{b_{2}}{1 + b_{1} + b_{2}}.}}$

A similar procedure can be used if a larger number of calibration runsis needed.

Notably, the transition from NCA to WCA detection system causes some ofthe collected fluorescence to be projected on the ‘dead’ insensitivespaces of the PMT, introducing additional loss in the efficiency of thedetector. It has been shown that in an exemplary system using PMT H7260in accordance with the present invention, the loss in fluorescence doesnot exceed 20% if cross-talk between adjacent channels stays below 30%(0.3).

The described cross-talk cancellation algorithm assumes that thephotodetection unit has strictly linear characteristic, i.e., itproduces the output response (in the case of the PMT module it is therate of shaped electric pulses) that is proportional to the intensity ofincident light. The characteristics of real world devices generally arenot strictly linear. In many cases a photodetection unit can becalibrated and the calibration results can be used to compensatenon-linearity in the experimental data. The compensation can beperformed in software or hardware. The algorithms for calibration andcompensation of non-linearity are described in detail in Gavrilov et al.After non-linearity compensation the data can be forwarded to thecross-talk cancellation algorithm of the present invention, which willnot introduce additional distortions in the data.

The NCA and WCA detection systems analyses assume that thecharacteristics of the PMT are known precisely and its non-linearity canbe perfectly compensated. The detailed description of the methods forcharacterization of single photon detectors and non-linearitycompensation is presented in Gavrilov et al. In analysis we also assumethat significant cross-talk exists only between the adjacent channelsand does not exceed 30%. The expressions are derived for the case whenthe number of lanes M is the same as the number of PMT channels N.

A software module may be implemented in accordance with an exemplaryembodiment of the present invention to automatically determine the lanecross-talk matrix based on calibration data. The software module mayprocess a series of calibration runs and produce a configuration file ofthe lane cross-talk matrix coefficients. A set of active lanes(containing sample) may be specified for each calibration run. Thecross-talk removal is performed separately on data obtained in each ofthe four fluorescence spectral bands. The procedure of estimation of thecross-talk matrix in every spectral band may include base-line removalin all 32 channels followed by peak detection.

The module may search for well resolved fluorescence peaks in the activechannels and corresponding cross-talk peaks in the neighboring channels.After non-linearity compensation, noise filtering, and base linesubtraction have preferably been performed, the peak heights in activeand neighboring channels may be determined. The cross-talk coefficientsare found as ratios of peak heights in neighboring channels to the peakheight in active channels. The coefficients may be further normalized.The determined cross talk matrix may then be applied for furtherprocessing of the sequencing data.

Experiments involving the cross-talk cancellation method of the presentinvention applied to processing of the experimental traces obtained fromInternal Lane Standard 600 (Promega Corporation) sequenced in the16-capillary array (Applied Biosystems Inc.) and detected using our32-channel DNA sequencing setup showed favorable results.

While there have been described what are believed to be the preferredembodiments of the present invention, those skilled in the art willrecognize that other and further changes and modifications may be madethereto without departing from the spirit of the invention, and it isintended to claim all such changes and modifications as fall within thetrue scope of the invention.

1. A method for reducing cross-talk in a multi-channel data acquisitionsystem, which system includes at least a multi-channel photodetector,comprising: determining a cross-talk matrix for the system; operatingthe system to detect data, and applying the cross-talk matrix to thedetected data to reduce or remove both optical and electrical channelcross-talk in the detected data.
 2. The method of claim 1 wherein thecross-talk matrix is determined theoretically using one or moremathematical formulas which characterize the operation of the system. 3.The method of claim 1 wherein the cross-talk matrix is determinedexperimentally by calibrating the system by applying known input dataand observing the resulting output data.
 4. The method of claim 1wherein the cross-talk matrix is determined experimentally bycalibrating one or more subcomponents of the system statically beforethe subcomponent is installed in the system.
 5. The method of claim 1wherein the cross-talk matrix is determined experimentally bycalibrating the system dynamically after the system is assembled.
 6. Themethod of claim 1 wherein the detected data is fluorescence radiation.7. The method of claim 1 wherein the data is detected by all channels ofthe system simultaneously.
 8. The method of claim 1 wherein the data isdetected by all channels of the system sequentially.
 9. A method forreducing cross-talk in a multi-channel data acquisition system, whichsystem includes at least a multi-channel photodetector, comprising:determining a cross-talk matrix for the system: operating the system todetect data, and applying the cross-talk matrix to the detected data toreduce or remove channel cross-talk in the detected data; wherein eachchannel of said multi-channel system detects and distinguishesfluorescence in more than one spectral band.
 10. A method for reducingcross-talk in a multi-channel data acquisition system, which systemincludes at least a multi-channel photodetector, comprising: determininga cross-talk matrix for the system; operating the system to detect data,and applying the cross-talk matrix to the detected data to reduce orremove channel cross-talk in the detected data; wherein each channel ofsaid multi-channel system detects and distinguishes fluorescence in morethan one spectral band; wherein the cross-talk reduction is individuallyperformed for each of the spectral bands.
 11. A method for reducingcross-talk in a multi-channel data acquisition system, which systemincludes at least a multi-channel photodetector, comprising: determininga cross-talk matrix for the system: operating the system to detect data,and applying the cross-talk matrix to the detected data to reduce orremove channel cross-talk in the detected data; wherein each channel ofsaid multi-channel system detects and distinguishes fluorescence in morethan one spectral band; wherein the cross-talk reduction is individuallyperformed for each of the spectral bands; wherein the cross-talkreduction is followed by color de-convolution of said fluorescence. 12.The method of claim 1 further comprising the step of applying anon-linearity compensation algorithm to the detected data.
 13. A systemfor reducing cross-talk in multi-channel data acquisition, comprising: amulti-channel photodetector; a processor, and a memory coupled to theprocessor and containing instructions which, when executed by theprocessor, configure the processor to perform steps comprising:determining a cross-talk matrix; operating the system to detect data,and applying the cross-talk matrix to the detected data to reduce bothoptical and electrical cross-talk in the detected data.
 14. The systemof claim 13 wherein the cross-talk matrix is determined theoreticallyusing one or more mathematical formulas which characterize the operationof the system.
 15. The system of claim 13 wherein the cross-talk matrixis determined experimentally by calibrating the system by applying knowninput data and observing the resulting output data.
 16. The system ofclaim 13 wherein the cross-talk matrix is determined experimentally bycalibrating one or more subcomponents of the system statically beforethe subcomponent is installed in the system.
 17. The system of claim 13wherein the cross-talk matrix is determined experimentally bycalibrating the system dynamically after the system is assembled.
 18. Asystem for multi-capillary data acquisition comprising; a light source;at least one multi capillary array positioned to receive light from thelight source and filled with a material which produces a usefulradiation caused by illumination with said light source; a device forseparating and identifying more than one spectral band of said usefulradiation; a multi channel photodetector positioned to receive saiduseful radiation from the multi capillary array; a processor configuredto receive data originating from the photodetector and a memory coupledto the processor and containing instructions which, when executed by theprocessor, configure the processor to perform steps comprising:determining a cross-talk matrix; operating the system to detectfluorescence data, and applying the cross-talk matrix to thefluorescence data to reduce both optical and electrical cross-talk inthe fluorescence data.
 19. The system of claim 18 wherein the cross-talkmatrix is determined theoretically using one or more mathematicalformulas which characterize the operation of the system.
 20. The systemof claim 18 wherein the cross-talk matrix is determined experimentallyby calibrating the system by applying known input data and observing theresulting output data.
 21. The system of claim 18 wherein the cross-talkmatrix is determined experimentally by calibrating one or moresubcomponents of the system statically before the subcomponent isinstalled in the system.
 22. The system of claim 18 wherein thecross-talk matrix is determined experimentally by calibrating the systemdynamically after the system is assembled.
 23. The system of claim 18wherein the data is detected by all channels of the systemsimultaneously.
 24. The system of claim 18 wherein the data is detectedby all channels of the system sequentially.
 25. The system of claim 24wherein the cross-talk reduction is individually performed for each ofthe spectral bands.
 26. The system of claim 18 wherein each channel ofsaid multi-channel system detects and distinguishes fluorescence in morethan one spectral band.
 27. The system of claim 26 wherein thecross-talk reduction is followed by color de-convolution of saidfluorescence.
 28. The system of claim 18 which perform multicolor DNAsequencing.
 29. A method for determining a cross-talk matrix for amulti-capillary data acquisition system, comprising: applying an inputsample to a first capillary; detecting fluorescence at a first channelwhich corresponds to the first capillary and at least a second channel;recording the detected fluorescence data; processing the recorded datafrom the first and the second channels to determine what fraction of thefluorescence from the input sample was received by the first channel andwhat fraction of the fluorescence from the input sample was received bythe second channel, and recording the determined fractions in a columnof the cross-talk matrix.
 30. The method of claim 29 further comprisingthe step of isolating at least a third channel from fluorescence emittedfrom at least a second capillary.
 31. The method of claim 30 wherein thefluorescence from the second capillary is isolated using a mask.
 32. Themethod of claim 29 where the input sample produces a fluorescence signalwith a specific signature which distinguishes this signal from otherphoto-signals in the data acquisition system.